This exploratory/developmental project is designed is to establish novel methodology as a research tool in the PI's laboratory to explore the neurobiological basis of intriguing preliminary findings that the phytocannabinoid cannabidiol (CBD) attenuates cocaine seeking with effects that significantly outlast treatment. A major factor contributing to the compulsive and chronically relapsing nature of cocaine addiction is drug desire elicited by environmental stimuli that have become conditioned to cocaine's subjective effects. In animals, the efficacy of these stimuli to elicit cocaine seeking perseverates over long periods of abstinence despite frequent exposure under non-reinforced conditions, reflective of the compulsive nature of cocaine addiction. In preliminary studies, CBD (the main non-psychoactive and non-addictive component of the cannabis sativa plant) significantly attenuated reinstatement in an animal model of perseverating, compulsive- like cocaine seeking, with effects that were still unabated six weeks after treatment termination. These findings suggest that CBD reverses neuroplasticity underlying cocaine craving and relapse beyond mere transient pharmacological amelioration of vulnerability to relapse. Insight into the mechanisms underlying these effects may therefore have major implications for treatment drug development and understanding of the neural and molecular basis of compulsive cocaine seeking. The objective of this proposal is to explore the neurobiological basis of CBD's lasting anti-reinstatement actions by exploiting recent advances in fluorescence-activated cell sorting (FACS) and associated methodologies. These advances, spearheaded by the Co-I, Dr. Hope, permit rapid high-throughput regionally specific identification (neural mapping) of Fos-expressing neurons that encode specific behaviors while at the same time providing RNA in a rapid and quantitative manner to permit characterization of molecular alterations in behaviorally activated neurons from single rats. The research plan is to extend the exploration of CBD's long-lasting effects on responsiveness to cocaine cues and ensuing cocaine seeking at the behavioral level, and to establish FACS and associated methodologies in the lab to identify brain sites and gene expression linked to the lasting attenuation of cocaine seeking by CBD. The results are expected to provide essential insight into neural and molecular targets through which CBD exerts its actions and to lay the foundations for subsequent full-scale projects ranging from systematic investigation of causal roles of identified neural targets and gene expression changes in mediating CBD's interference with cocaine seeking, to utilization of FACS for the identification of key genes and their epigenetic regulatory mechanisms responsible for the persistence of CBD's actions, and thereby to reveal novel therapeutic targets for relapse prevention.